The ATPase, kinesin, which transports particles towards the plus-ends of microtubules (MTs) in vitro, is thought to participate in a variety of important cellular and developmental processes including mitosis, nuclear migrations, vesicle trafficking, and organizing intracellular membranes. The structural and functional characterization of kinesin is progressing rapidly, but little is known about the regulation of kinesin activity. We previously purified and characterized kinesin from sea urchin eggs and embryos, and found that it consists of 2 mol 130Kd heavy chain (HC) : 1 mol 84Kd light chain (LC) : 1 mol 78Kd LC. Recently we have observed that the HC and LCs can be phosphorylated in vivo and in vitro, and here we propose a detailed characterization of these phosphorylation reactions, together with a study of their effects on kinesin-driven intracellular transport. We will use phosphoamino acid analysis, phosphopeptide mapping and radiosequencing methods to determine the number, location and sequence of the phosphorylation sites on kinesin HC and LC. Phosphopeptide antibodies will be raised for use in monitoring the abundance and distribution of phosphorylated kinesin in cells and subcellular fractions. We will use kinesin or kinesin peptides as substrates for characterizing kinesin phosphorylation-dephosphorylation reactions, in assays for purifying kinesin kinases and phosphatases from sea urchin cytosol, and comparing them to previously-characterized enzymes (a number of which will also be screened for kinesin kinase or phosphatase activity). If this work reveals that a kinesin kinase or phosphatase is a novel enzyme we will characterize its localization, as well as its structural and biochemical properties. To investigate the effects of phosphorylation on the intracellular distribution of kinesin, subcellular fractionation and immunocytochemistry will be performed under conditions that enhance or inhibit kinesin phosphorylation. Most important, we will compare the activities of phosphorylated and nonphosphorylated kinesin preparations using motility assays, membrane binding/transport assays, and MT-activated ATPase assays. Thus we hope to learn if HC and/or LC phosphorylation controls (1) the mechanochemical activity of kinesin and (2) the attachment of kinesin to its transported cargo.